Sophorolipids are used in, for example, cosmetology, as antiradical-type agents and anti-elastic agents (WO 95/34282). They can also be used as co-surfactants in a process for clearing soil of pollution (EP-A 605 308).
It was mentioned in U.S. Pat. Nos. 3,205,150, 3,312,684 and EP-B 516803 that a quantity of sophorolipids was produced by a fermentation process that employed a Candida bombicola culture.
Sophorolipids are considered to be a mixture of compounds represented by formulas (1) and (2), in which R.sup.1 represents hydrogen or the acetyl group and R.sup.2 represents hydrogen or an alkyl group that contains 1 to 9 carbon atoms, or else R.sup.2 represents hydrogen or a methyl group, when R.sup.3 is an unsaturated chain that contains hydrocarbon with 13 or 17 carbon atoms.
These compounds can be used as cleaning agents and as emulsifiers, and they exhibit excellent hygroscopic properties and hydrophilic properties due to the sophorose portion of each of these molecules and hydrophobic properties that come from the fatty acid portions.
The preparation of sophorolipids is generally done in the presence of a substrate as described in U.S. Pat. No. 3,205,150. For example, this substrate can be made of hydrocarbons, saturated or unsaturated fatty acids, acid esters including glycerides, and vegetable oils such as soybean oil.
The substrate feed consists in ensuring, at intervals of about 12 to 24 hours, the discontinuous (batch) injection of a quantity of about 2% by weight relative to the initial reaction volume for each addition. The substrate feed is more advantageously carried out continuously (fed batch), as in the case of the European patent from applicant EP-B 516803. ##STR1## Structure of the sophorolipids: (1): acid forms
(2): lactone forms
The discontinuous fermentation processes with the discontinuous (batch) feed of substrate or continuous (fed batch) feed of substrate have the classic drawbacks of discontinuous fermentation. Thus, the repeated use on an industrial scale of a series of discontinuous operations (by batch or by fed batch) means much down time in the operation of installations for cleaning and sterilization of the fermenter, which have to be repeated between each cultivation. Likewise, the propagation of the productive stock from its state of preservation in a gelose-treated tube, for example, to the production fermenter has to be done each time via an inoculation chain which involves the successive cultivation of the microorganism in fermenters of increasing sizes. This precultivation operation can take several days.
To avoid these drawbacks and constraints, technologists usually resort to continuous cultivation, in which the reactor is fed with culture medium and substrate at equivalent flow rates and the fermented wort is drawn off at an equivalent flow rate. The reaction volume is then constant. Routinely, continuous cultivation is carried out according to the chemostat method (Wang et al. 1979. Continuous Culture. In Fermentation and Enzyme Technology, John Wiley & Sons (eds), pp. 98-137). A nutrient for the culture medium is present in a limiting quantity, so that it is possible to monitor the steady-state concentration of the microorganism in the fermenter and to obtain stable operation of the system. When it is desired to produce metabolites (such as ethanol, for example), two or more fermenters that are arranged in series (bi-stage or multi-stage system) are generally used. In the first, the growth of the microorganism (first fermentation stage) is carried out with a medium that contains a limiting nutritive element (nitrogen, phosphorus), while the excretion of metabolites is carried out in the second reactor into which the fermentation wort from the first stage is admitted. Completely unexpectedly, it was observed that the stock that was placed under these production conditions progressively and quickly lost its excretion properties and that productivity was very inferior to that observed with fed batch.
Also studied, in Chem. Abstr., Vol. 122, No. 15, Apr. 10, 1995, No. 185450, was a fermentation process pertaining to the production of sophorolipids, so-called "self cycling fermentation (SCF)" during which, at the end of a period of time that corresponds to the growth period of the stock, half the fermentation wort obtained, which is replaced with fresh medium, is drawn off (see J. Ferment. Bio Eng. 1995, 79(2), p. 146, col. 1, 1.11 before the end, combined with the document Chem. Abstr.).
It is recommended that the cycle length after the growth period of the stock (doubling time) be extended by about 2 hours, so that each new cycle hardly exceeds 6 hours. With this approach, to be sure, the production capability of the stock is maintained, but the production of sophorolipids is very low, and it is very difficult to recover them short of extracting them with a solvent, which complicates the process and makes it even more expensive.
Other documents, such as Chem. Abstr., Vol. 119, No. 3, Jul. 19, 1993, No. 26744, Patent Applications FR-A.2 670 798 and FR-A. 2 692 593 describe a process for the production of sophorolipids by fed batch that includes a fermentation cycle.
Finally, Patent Applications DD-A-252 002 and DD-A-254 959 relate to cyclic fermentation processes for the production of proteins, steroids, and antibiotics by fungi and bacteria, whereby these processes neither describe nor suggest the production from yeast of glycolipids, which are products that are very different from those mentioned above.